Production and Characterization of Nutritious Peanut Butter Enhanced with Orange Fleshed Sweet Potato

Crimson Publishers Research Article Wings to the Research

Mulindwa J1, Kaaya NA1, Tumuhimbise G1 and Naveen Puppala2* 1School of Food Technology, Nutrition and Bioengineering, Makerere University, P.O.BOX 7062, Kampala, Uganda. ISSN: 2640-9208 2New Mexico State University, Agricultural Science Center at Clovis, New Mexico 88101, USA.

Abstract Peanuts worldwide are popular for their nutritional quality and commercial potential. Their consumption

(OFSP)in Uganda is equally is high highand secondin the country after common and this beans too offers thus makingpotential them to fortify a suitable peanut food butter for fortification for increased to intakefight the of vitaminincreasing A. Thevitamin objective A deficiency of this study in the was country. to investigate Consumption the potential of orange of producing fleshed sweet a nutritious potato peanut butter, with high shelf-life. An OFSP ratios of 0% (Control), 5% (Treatment 1), 10% (Treatment 2) and 15% (Treatment 3) were mixed with peanut butter. The product was assessed for proximate composition using AOAC methods and sensory qualities. The shelf-life of product was also established by determining the fat quality, beta-carotene retention and microbial quality. Fortifying peanut butter with *1Corresponding author: Naveen Puppala, Department of Agricultural from 2.96 to 25.51% and, beta-carotene from 244 to 1388µg 100g-1. In all treatments, the control had the Science Center at Clovis, USA OFSP significantly increased the protein content from 20.47 to 27.76%, fat from 30.8 to 32.4%, sugars

November 1, 2019 Submission: lowest amount of nutrient, while OFSP that-1 wasg which fortified could with meet 15% the peanut daily butterWorld had Health the highestOrganization levels Published: December 04, 2019 of the nutrient. When OFSP was fortified with 10-1 and 15% peanut butter it resulted in higher retention of β-carotene between 400 to 600μg 100 below(WHO) 0.9mg recommendations KOH-1 and peroxide of 350 valueto 500μg (PV) 100 belowg. After 4mEq storing kg-1 respectively. the product There for five was months, a strong OFSP negative that Volume 4 - Issue 4 was fortified with 10 and 15% peanut butter had good fat quality as reflected by the low acid value (AV) butter. All peanut butter samples were free of dangerous levels of microbes. The peanut butter treated How to cite this article: Mulindwa withcorrelation OFSP had(r=0.049; acceptable p˂0.05) sensory between score peroxide of 6-7 onformation the scale and of the1 to amount 9. The resultsof β-carotene suggest in that the peanut J, Kaaya NA, Tumuhimbise G, Naveen Puppala. Production and Characterization children. of Nutritious Peanut Butter Enhanced with butter fortified at 15% OFSP had greater shelf-life and meet the vitamin A requirements of school going Orange Fleshed Sweet Potato. Nov Tech Keywords: Nutri Food Sci. 4(4).NTNF.000593.2019. DOI: 10.31031/NTNF.2019.04.000593 Peanut butter; OFSP; β-carotene; Fat quality

Copyright@ Naveen Puppala. This Introduction article is distributed under the terms of Peanuts (Arachis hypogaea L) are popular worldwide because of their value as plant protein the Creative Commons Attribution 4.0 International License, which permits source (23-35%) and fat (45-52%) [1]. The peanuts possess high nutritional and commercial unrestricted use and redistribution value due to the presence of fatty acids, protein, carbohydrates, minerals and vitamins [2,3]. provided that the original author and Globally, peanut consumption is relatively high and is consumed either as roasted, cooked source are credited. or as peanut butter [4]. In Uganda, peanuts rank second with annual production of 210,000 tons in shell after common beans (Phaseolus vulgaris; FAO, 2017). Peanuts are potential food

source for fortification since they are consumed widely in Uganda in various forms as sauce, children and pregnant women has been reported at a rate of 19% to 20% respectively [5]. peanut butter and paste. In Uganda increasing prevalence of vitamin A deficiency amongst This situation along with limited access to nutritious foods adversely affects the wellbeing of

children and adults. Consumption of peanut butter fortified with vitamin A is considered as a wayPeanut to reduce butter vitamin is a A semi-perishabledeficiency [6,7]. product with prolonged shelf life due to its low moisture content [8]. Peanut products stored in ambient conditions are exposed to sunlight. The heat accumulated during storage accelerates rancidity [8-10]. The rancid peanut butter is

Novel Techniques in Nutrition and Food Science 356 NTNF.000593. 4(4).2019 357

-carotene (C0), 5% (Treatment 1), 10% (Treatment 2) and 15% (Treatment is a powerful antioxidant that provide protection against oxidative 3). Varying ratios were used to increase the concentration of unfit for consumption because of off flavors [11,12]. The β processes in food systems [13,14]. The antioxidant activity -carotene and putting into consideration the effect of solids on

β Orange Fleshed Sweet Potato (OFSP), one of the major sources OFSP pulp because of the deteriorative effect that pulp can impose of β-carotene is attributed to their polyene frameworks [15]. the quality of peanut butter [23]. The OFSP flour was chosen over of beta-carotene is widely grown and consumed in Uganda [16]. on the product due to high moisture content. Mixing was done In the year 1995, researchers recognized the potential of OFSP using a dough mixer (Type: 94/R10; No. 21602) for 15 minutes to achieve uniform and consistence mixture, and 0.7% of triglyceride Saharan Africa using integrated agriculture-nutrition approach stabilizer was added. The OFSP peanut butter and control sample varieties to address widespread vitamin A deficiency in Sub were packed in food grade plastic jars. which the body converts into vitamin A [17]. Through the multi- [17]. Use of OFSP is a rich plant-based source of β-carotene, Chemical analyses partner initiative, OFSP was launched in Uganda headed by Harvest-Plus. Various Non-Government Organizations (NGO), The samples packaged in food grade containers were delivered Volunteer Efforts for Development Concerns (VEDCO), Farming to Makerere University chemistry laboratory for proximate analysis for Food and Development Program-Eastern Uganda (FFDP-EU) and National Agricultural Research Organization (NARO) have and shelf stability (acid value, peroxide value, -carotene retention (Moisture content, protein, fat, sucrose, fiber and beta-carotene), since disseminated OFSP in Uganda to create awareness and have and microbial quality) studies. β released varieties such as Ejumula, Vita, and Kabode among others; Moisture and value addition for increased consumption [18]. Research has shown that OFSP has the potential to improve the vitamin A status About 3g of each sample was weighted in the dry dishes and of individuals [19,20]. Study by Jaarsveld et al. (2005) showed that, weight recorded. The dishes with the sample were put in the oven and dried for about 6 hours at temperature of 95 °C. The dishes stores amongst the school children who were fed on OFSP. Product were then cooled in a desiccator and weights recorded and percent there was a 10% significant improvement in Vitamin A that liver diversity can be a driver to its increased consumption especially moisture determined, amongst the children. This study therefore aimed at production of % M. C=( Wt of sample before drying−× wt of sample after drying) /( wt of sample before drying ) 100

% M. C=( Wt of sample before drying−× wt of sample after drying) /( wt of sample before drying ) 100 that could be used by school-going children. a shelf stable, high nutritious OFSP-fortified peanut butter product Materials and Methods Protein Materials Crude protein content of samples was determined using the standard Kjeldahl method [24]. About 0.2g of each sample was Twenty kilograms of peanut (Valencia variety) were obtained digested using 5ml concentrated sulphuric acid and Kjeldahl tablets from the National Semi-Arid Resources Research Institute, Soroti, Uganda. Triglyceride stabilizer was purchased from Dansico minutes, heated rapidly after stabilization for 2 hours then left to as catalysts. The sample solution was heated slowly for the first 6 Company, United States of America (USA). Two hundred (200) cool. The digest was quantitatively transferred to a 50ml volumetric purchased from VEDCO Uganda at maturity age of 4 months homogenize the solution. The sample distillate was prepared kg of Orange fleshed sweet potato roots (Kabode variety) were flask and made to volume with distilled water, then shaken to when roots have attained dark orange color and expected to by pipetting 10ml aliquots of the digest in a Markham still (Foss, contain highest -carotene content. Chemicals and reagents used Tecator, Britain), 20 ml of 40% sodium hydroxide was introduced in laboratory analysis were obtained from Westford laboratory, β into the distillation chamber and distillation was allowed to Kampala, Uganda. proceed for about 4 minutes. The distillate was collected into the Preparation of OFSP peanut butter (bromocresol green and methyl red); the end point was marked conical flask containing 10 ml boric acid (4%) and mixed indicators Peanut butter was produced following [21], with some by color change back to the original brown color. The blank titer was subtracted from the sample titer and the total crude protein were selected, cleaned using a 2-step wise cleaning method; 1) modifications to suit the available technology. Peanut kernels determined using the equation below: dry cleaning where sorting is done, and 2) wet cleaning where the %N22 in sample g/100 g=( 0.05 MHCL ×× titre14 atomic wt of N× 6.25 factor × 100) /( 1000 ×Sample wt) kernels were washed to remove dust on the surfaces. The peanut kernels were then roasted %inN the22 in sampleelectrical g/100 oven g (Model:=( 0.05 MHCL GU-6) ×× for titre 14 atomic wt of N× 6.25 factor × 100) /( 1000 ×Sample wt) 25 minutes at a temperature of 140 ° C, then cooled for 5 minutes and test was removed to ease sorting of seeds by color to reduce the Note: (Titre X NHCL/1000 = No. of mole NH3) Dietary fiber were ground using a blade grinder (Capacitor Start Motor; type: incidences of aflatoxin infection [22]. Peanuts that passed sorting, YC112M-2; HP 248A) till a smooth peanut butter was formed. The Fibre (ADF) standard method [24]. One gram of each sample Dietary fiber was determined on the basis of Acid Detergent OFSP flour was added to the smooth peanut butter in ratios of 0% was weighed and mixed in 100ml of acid detergent fiber (28ml

concentrated sulphuric acid and 20g cetyl trimethyl ammonium Shelf-life of OFSP peanut butter under different conditions analyzer (Labconco Corporation, Kansascity, Missouri 64132. Serial Bromide) solution. The solution was boiled for 1 hour on the fiber The peanut butter with added OFSP and control sample crucible. The crucible was dried in the oven for 30 minutes and No. 246719) and then filtered through a pre-weighed glass sintered environment of peanut butter and then analyzed for quality changes were stored under ambient conditions that reflected the retail over a period of 5 months. Fat quality (acid value and peroxide using the formula below: cooled in the desiccator before weighing. The fiber was determined value), -carotene retention and microbial quality (microorganisms % dietary fibre= ( fibre extracted) /( sample wt)×100 of interest were E. coli, S.aureus, yeasts and moulds)was determined β every after a month. Fat About 3g of sample was weighed into a thimble in triplicates. Fat quality The thimbles and their contents were placed into 50ml of Acid value (AV): Acid value of treatments and control sample petroleum ether (PE) in a beaker assembled in the Soxhlet system. was determined [24] by weighing 3g of each sample into 100ml The fat in the sample was extracted using PE, by boiling at 115 ° C for 20 minutes and then rinsed for 45 minutes. The beakers were ether, v/v) with phenolphthalein were added to the sample in the transferred to the oven to evaporate off the PE and other water- conical flask. Solvent mixture (50ml; neutral 95% ethanol: diethyl an interval of 3 minutes to ensure that the free fatty acids in the flask. The mixture was allowed to stand for 20 minutes shaking at in the desiccator to room temperature and weights taken. sample dissolve into the solvent. The supernatant was decanted off soluble material for 30 minutes at 90 ᵒC. The beakers were cooled % Crude fat= ( Wt of fat extracted) /( Sample wt in g ) ×100 and was titrated with standard sodium hydroxide solution to the pink endpoint (the pink color persisting for at least 10 seconds). Sugar The acid value was expressed as percentage. Total sugars were determined by hot water extraction method 56.2**NV Acid Value = (AOAC, 2002). One gram of each sample of peanut butter was M accurately weighed into 250ml beakers to which 1ml lead acetate Where; was added followed by 70ml of hot water. The beakers with the contents were then placed on a hot water bath at 80 °C and heated V is the number of ml of NaOH solution used for 1 hour. To the cooled sample solution, half a spatula of sodium N is the exact normality, and bicarbonate was added to precipitate all the excess lead acetate. M is the mass in g of the sample quantitatively and shaken to mix well. A portion of the sample was The sample was then transferred to 100ml volumetric flask poured into test tubes and centrifuged at 700rpm for 5 minutes. Peroxide Value (PV) The peroxide value was determined [24] by weighing 5g of Five ml of the clear solution of the sample, 1 ml of concentrated sample into a beaker and mixed thoroughly in a 30ml mixture of sulphuric acid and 20ml of distilled water were added to 100ml 3:2 glacial acetic acid and chloroform solution by vigorous shaking. Saturated potassium iodide solution (0.5ml) was added to the solution was neutralized with sodium bicarbonate and transferred conical flasks and then heated to boiling for 10 minutes. The cooled mixture, as a result of which iodine was liberated due to reaction with the peroxide. This was then titrated against a standard distilled water and mixed. To develop the color, 1ml of sample was quantitatively to 50ml volumetric flask and made to volume with solution of sodium thiosulphate, using starch solution as indicator. added followed by 1ml of phenol (5%) and 5ml of concentrated The procedure was repeated to determine the titration value for a sulphuric acid to a clean test tube and mixed well. The absorbance blank sample. PV was calculated as below: of the solution was read off at 470nm. PV. = S − B* N *1000 / Sample weight mEq/ kg β-carotene (( ) ) ( ) ( ) Where; Following Rodriguez et al. [18], three g of peanut butter was weighed in the mortar. Using 50ml of cold acetone, the sample was S=Titration value of the sample (ml) ground to extract the carotenoids. Experiment was repeated until B=Titration value of the blank sample (ml)

N= Normality of the Sodium Thiosulphate solution= 0.01N the sample was colorless, and then mixture was filtered through a remove the acetone residue, the mixture was washed in a 500ml funnel. About 30ml of petroleum ether were added to filtrate. To Sample Weight=5gm separator funnel using 300ml of distilled water, this was repeated three times. Petroleum ether (PE) phase was collected in a 50ml Microbial Analysis sulfate to remove residual water. Absorbance of beta-carotene was Staphylococcus auerus volumetric flask through a funnel containing 15g anhydrous sodium read at 450nm using a spectrophotometry. Ten grams of peanut butter sample was added into sterile bottles having 90ml peptone water. After thoroughly mixing, the

Nov Tech Nutri Food Sci Copyright © Naveen Puppala NTNF.000593. 4(4).2019 359 sample was serially diluted up to 10-6. Twenty ml Baird parker using 9-point Hedonic Scale, where 1=dislike extremely, and 9=like agar (BPA) was poured on Petri-dishes and left to set at room extremely [25]. Data analysis temperature. After complete solidification, the plates were inverted samples (0.1ml) of dilutions 10-1 and 10-2 were surface spread Data for sensory evaluation was analyzed using SPSS [26]. Data to avoid dripping of condensed water on solidified agar. Duplicate on proximate analysis and shelf-life of the peanut butter sample plates were incubated at 37 °C for 3 days. Enumeration was done were tabulated and means subjected to ANOVA using Genstat on the solidified plated petri-dishes using sterile glass rod. The considering spreaders and clusters as a single colony (ISO 21527-2) 13th

Yeasts and moulds Edition). The means were separated using LSD (P≤0.05) to determine significant differences. Yeasts and moulds count were made by adding 10g of peanut Results and Discussion butter sample into sterile bottles having 90ml peptone water. After Chemistry thoroughly mixing, the sample was serially diluted up to 10-6. Although the moisture content of the control (C0) was

Acidified agar (15-20 ml) was poured on Petri dishes and left to set significantly lower than the treatment samples (P< 0.05), the at room temperature. After complete solidification, the plates were agar. Duplicate samples (0.1 ml) of 10-1 and 10-2 dilutions were moisture content of the latter did not differ significantly implying inverted to avoid dripping of condensed water onto the solidified that increased amount of OFSP have no influence on the moisture glass rod. The plates were incubated at 30 ° C for 3 days in upright content of the fortified peanut butter. The moisture content of the surface spread on the solidified plated petri-dishes using sterile McDaniel et al., 2012 who reported that peanuts have moisture position because yeasts and molds grow upwards. Enumeration control sample was 1.89% which is in agreement with findings of was done considering spreading colonies and clusters as a single colony (ISO 21527-2) content between 1.4 to 2%. Fiber content increased significantly (P<0.05) with an increase in the ratio of added OFSP flour to peanut Coliforms (E-coli) butter. The control sample had the least fiber content, followed samples with increased ratio of OFSP could be due to relatively high Ten grams of peanut butter sample were added into sterile by treatment 1, 2, and 3. The increase in the fiber content of the test bottles having 90ml peptone water. After thoroughly mixing, 3% [27]. the sample was serially diluted up to 10-6. Dilutions of 10-1 and 10-2 fiber content of OFSP which is reported to be in the range of 1.8 to were taken in duplicate samples (1ml) and pour plated using 20ml The results showed that addition of OFSP to peanut butter does of violet red bile agar. After thoroughly mixing, the plated sample was allowed to solidify and then incubated at 37 ° C for 24 hours. The fat content of the control and treatments ranged between 30.83 Counts were made considering the purplish red colonies as coliform not significantly affect the fat content of the peanut butter (Table 1). colonies and clusters as single colonies (ISO 4832). samples. The control sample (32.45%) and treatment 1 (32.53%) to 32.45% though there was no significant difference among the had the highest fat content while treatment 3 (30.83%) had the least Assessing acceptability of OFSP peanut butter Fifty consumer panelists were recruited from the School amount. The findings also show that, the amount of fat decreased of Food Technology, Nutrition and Bioengineering, Makerere current study showed that the fat content of the peanut butter was University. The panelists were briefed before the start of session. with increasing ratio of OFSP flour added to the peanut butter. The also reported peanut butter fat content of 32% in the peanut butter. between 32-30%, this is in agreement with the findings of [28] who to each panelist. Samples were evaluated in the order of appearance However, others reported higher fat content between 49 to 51% Four samples from the five treatment combinations were presented on the ballot. Panelists were asked to place a spoonful of peanut [29-31]. This variation in fat content could be due to differences in butter on plain bread to evaluate the spread ability and consistency. They were also asked to rinse their mouths with water between fat 0.41% [32] and this could explain why there was decrease in fat samples. The samples were evaluated and ranked by the panelists agro-ecology and varietal differences [21]. OFSP flour is devoid of

content of treatments with high ratio of OFSP flour. forTable color, 1: flavor, Proximate spread analysisability, consistency for peanut and butter overall samples. acceptability

Moisture (%) Fiber (%) Fat (%) Sugar (%) Β-carotene (µg/100g) Protein (%) Control 1.89±0.02a 5.31±0.07a 32.45±0.1a 2.96±0.61a 244±11.60a 27.76±0.76a Treatment 1 2.18±0.00b 5.61±0.06ab 32.53±0.2a 9.19±0.02b 795±111.50b 25.79±0.34b

Treatment 2 2.14±0.06b 5.87±0.08b 31.12±0.2a 16.45±3.3c 1041±36.80c 24.36±0.55b

Treatment 3 2.13±0.00b 6.25±0.14c 30.83±0.58a 25.51±0.11ad 1388±0.70d 20.47±0.08c

Values are means±standard deviations. Means followed by the same letter in the same column are not significantly different (p>0.05).

Note: Control (0% OFSP), treatment 1 (5% OFSP flour), treatment 2 (10%OFSP flour) and treatment 3 (15% OFSP flour).

The sugar and of OFSP increased, the protein content of peanut butter reduced

β-carotene contents in the study significantly with what was reported by Shakerardekani et al. [30]; Riveros et al. increased with increasing addition of OFSP flour in peanut butter, significantly. The results obtained in the study are in agreement [31] and Singh et al. [37], who reported protein content in peanut implying that the more OFSP flour used, the more sugar and butter in the range of 22 to 30%. According to Low et al., 2010, β-carotene content of the peanut butter. The control sample had 100g-1 respectively while Treatment 3 had the highest levels, over OFSP has a low protein value of 0.016% and this could explain why the least content of sugar and β-carotene of 2.96% and 244μg (1388µg 100g-1) respectively. The results also show that treatment increased substitution ratio of OFSP. eight times and five times of sugar (25.51%) and beta-carotene there was significant decrease in protein content of product with 2 had higher sugar and -carotene content than Treatment 1, and Fat quality -carotene β than control. The sugar content of the peanut butter was 2.96% Fat quality is very important as far as storage of peanut butter both treatments had significantly greater sugar and β which was in agreement with the literature as stated by Settaluri is concerned because it affects shelf-life due to oil susceptibility et al. [2]. Sweet potatoes have a relatively high sugar content, and to rancidity [30,31]. Rancidity is often used as an indicator of the stability and edibility of oils [38-40]. increased percentage of sugars. this explains why increase in its concentration led to significantly Acid value (AV) According to study done by King et al. [33], he reported that Changes in the AV of control (C0), Treatment 1 (5% OFSP), peanuts contain around 3µg 100g-1 -carotene, while [34] reported Treatment 2 (10% OFSP), and Treatment 3 (15% OFSP) (Figure -1 and Pattee et al. [35] β 1) showed a gradual increase as the OFSP ratios and storage time reported -carotene of 60µg 100g-1 β-carotene content in peanuts of 15.23μg 100g increased. Acid Value in 5th to the results of the current study, and this natural variation may be β . All the findings are in contrary in all samples, with control (C0) showing 109% increase followed explained by the geographical and varietal differences. On addition month of storage increased significantly by 91% in treatment 1, 81% in treatment 2, and 76% in treatment of OFSP to peanut butter, beta-carotene increased to values that could meet the World Health Organization [36] daily recommended 1.08 milli-grams of potassium hydroxide per gram of fat (mgKOHg-1) in takes of 350 to 500µg 100g-1 for children between 5 and 16 years. 3. By the fifth month, AV of control and treatment 1 had increased to and 1.004mg KOHg-1 respectively. Treatment 2 and treatment 3 were still below 1mg KOHg-1. Kirk et al. [40] stated that when acid with control having the highest protein content (27.76%), followed value is in the range of 1 to 1.5mg KOHg-1, rancidity is detected by The protein content significantly ranged from 20.47 to 27.76%, by treatment 1 (25.79%), then treatment 2 (24.36%) and lastly sensory tests. treatment 3 (20.47%). The results reflect that, as substitution ratio

Figure 1: Changes in AV concentration of the OFSP peanut butter and control sample with storage time. Line labeled ** shows the limit beyond which acidity of oils can start to affect sensory properties. Control sample (C0), Treatment 1(5% OFSP), Treatment 2 (10% OFSP) and Treatment 3 (15% OFSP).

The AV represents the amount of the free fatty acids present described by Bendini et al. [38]. The increase is triggered by in food sample and is determined by measuring the number of exposure of lipase and other lipolytic materials to atmospheric milligrams of potassium hydroxide required to neutralize the free oxygen after peanut crushing [42]. Light and heat also accelerate fatty acids in 1g of the sample. The AV also shows the extent to the breakdown and decomposition of fats to free fatty acids [43]. which the glycerides in the oil have been decomposed by lipase Since the peanut butter samples were stored at ambient conditions, [40]. Thus, every increase in the potassium hydroxide shows the there was a possibility of exposure to elevated temperatures and presence of more free fatty acids and also indicates lipase activity light conditions during storage, which could have led to increased on fats [41]. The free fatty acids increase with storage time as formation of free fatty acids.

Peroxide value (PV) of OFSP peanut butter samples 19.62meq kg-1 (Figure 2). The results also show that, treatments with low OFSP ratio had high rate of increase in the peroxide value. At peroxide value of 10meq kg-1, oxidation reactions are initiated, 3 did not register any peroxide unlike treatment 1which recorded From the first to the third month of storage, the treatment 2 and some peroxides. The control sample (C0) had peroxides formed in and rancid flavors may start to be noticed. The results however storage, all the samples had registered some levels of peroxides showed that, for the first four months of storage PV was not high the second month of storage. During the fourth and fifth month of to cause rancidity unlike in the fifth month where the PV for C0 significantly increased above the limit. but with C0 registering significantly high increase to a value of

Figure 2: Changes in PV with storage time for the different peanut butter with added OFSP and control sample. Line labeled** indicates the induction period beyond which peroxide formation accelerates rapidly and development of off flavors. Control sample (C0), Treatment 1 (5% OFSP), Treatment 2(10% OFSP) and Treatment 3 (15% OFSP).

PV is an indicator of the initial stages of oxidative change in food 19m Eq kg-1 of fat with C0 (19m Eq kg-1) having the highest and [44]. This method utilizes the principle of ferric ion complexion Treatment 3 the least PV (2.5m Eq kg-1). Therefore, the OFSP peanut where hydrogen peroxide (ROOH) is reduced with Fe2+ leading to butter had not yet attained the values necessary to produce the formation of Fe3+ complexes [41]. The concentration of peroxides as represented by the PV is useful in assessing the extent to which rancidThe flavors presence during of carotenoids the five months in OFSP of storage. can inhibit the formation spoilage has advanced. The report by Azhar et al. [45] indicated of peroxides. Amongst the carotenoids, -carotene has a higher that PV increased with storage time which is in agreement with the potent for peroxides, which involves formation of hydrogen radical current study which showed that PVs of the samples increased with β abstraction (ROO-CAR) complex, thus inhibiting utilization of the increasing storage time. Mailer et al. [46] also claimed that more free radicals by oxygen [15]. This may explain the reduced rates oxidation occurs in lipids with prolonged time of storage. When the concentration of peroxides reaches an induction point (10m peanuts have naturally occurring phytochemicals like tocopherols Eq kg-1), complex chemical changes occur, and volatile products of peroxide formation in samples with OFSP flour. Furthermore, and polyphenolics; these also play a role in slowing or preventing are formed that are mainly the rancid taste and odour [38]. In the lipid oxidation due to their anti-oxidative nature [41,47]. current study, the PV for the different samples was between 2.5-

Relationship of PV and AV with OFSP levels and storage time

Table 2: Correlation of PV with independent variable AV, storage time and OFSP ratio.

Parameter PV OFSP ratio Storage time (months) AV

PV 1

OFSP ratio -0.4971* 1

Storage time (months) 0.5852* 0 1

AV 0.7580* -0.1847 0.8955* 1

R2=68.9; Values with * have a significant positive or negative relationship at P≤0.05

There was no linear relationship between AV and OFSP ratio (r= β-carotene retention of treatment samples with storage time

-0.1847, P≥0.05) (Table 2). However, a strong positive relationship In all the samples, between AV and time of storage (r=0.8955, P≤0.05) was detected. PV storage time increased (Figure 3). The control sample (C0) had associated with OFSP ratio and storage time, respectively (Table2). β-carotene significantly reduced as the was significantly negatively (r=-0.4971) and positively (r=0.5852) the least with storage time. Treatment 3 with highest level of -carotene positively (r= 0.758). The negative relationship between PV and β-carotene which also significantly kept on reducing The correlations further show that AV significantly affected PV -1) in the 1st month of storage and it had reduced to OFSP indicates that OFSP was resisting the formation of peroxides. β 580.6µg 100g-1 in the 5th month. The results further show that, the This may be because -carotene contained in OFSP reacts with fat (1388.2μg 100g reduction in -carotene was proportion to the amount present in the radical to form a stable radical which does not quickly react with β samples. Treatment 2 and 3 which had high values, also registered oxygen [48]. Antioxidants terminate the free radical intermediates, β by being oxidized themselves, thus acting as reducing agents [48,49]. a significantly high loss with storage. However, at the end of the fifth month of storage, treatment 2 and 3 still had considerably high β-carotene levels compared to treatment 1 and control (C0).

Figure 3: Changes in β-carotene with storage time for the different peanut butter with added OFSP and control sample. Control sample (C0), Treatment 1: (5% OFSP flour), Treatment 2: (10%OFSP flour) and Treatment 3: (15% OFSP flour).

The losses in -carotene over time may be due to exposure of peanut butter samples to light during storage as -carotene is storage time and PV were negatively correlated (r=-0.5483 and; r=- β correlated with storage time, OFSP flour and PV value while both sensitive to heat and light [50]. The processing procedures and time 0.5852) with the -carotene retention, respectively. On the other β also expose hand, there was a strong positive correlation observed between β losses as noted by Bechoff et al. [51] and Wheatley [52]. In addition, OFSP ratio and the β-carotene to oxygen which may further influence the indicates that a decrease in -carotene during storage is natural β-carotene (r=0.7547, P≤0.05). The literature analysis may have introduced variability in the results obtained as the difficulty in complete extraction of the carotenoids during β it was also noted by Bengtsson et al. [16]. Despite the fact that there correlation was noted between beta-carotene and storage time [51]. This was also reflected in the study as a strong negative -carotene during storage, the quantities -carotene can be addressed retained by the Treatments 2 and 3 were high compared to the by increasing the amount added to the food. The current study was significant loss in β (r=0.5483, P≤0.05). The decrease in β showed that -carotene content correlates positively with the OFSP can contribute some level of -carotene o the daily -carotene control sample. Thus, peanut butter fortified with 10% and 15% β requirements. β β amount of OFSP flour added in the sample (r=0.7547, P≤0.05) Relationship between β-carotene with storage time, indicating that an increase in the OFSP flour increased positively OFSP ratio and PV units the level of β-carotene. These findings agree with Bechoff et al. [12] in foods increases the -carotene content. Results in Table 3 show that and Bengtsson et al. [16] who reported that more OFSP flour added β β-carotene was significantly

Table 3: Correlation of B-carotene with other independent variables.

Parameter β-carotene OFSP ratio Storage time (months) PV -carotene 1

βOFSP ratio 0.7547* 1 Storage time (months) -0.5483* 0 1 PV -0.5843* -0.4971* 0.5852* 1

R2=89.2; values with *have a significant positive or negative relationship (P≤0.05) -carotene content, is Changes in microbial quality of OFSP peanut butter during oxidation. Since -carotene plays an anti-oxidative role, the storage Among other factors that influence β increasing PVs of the peanut butter samples negatively affected β The presence of microbes such as Escherichia coli, the retention of -carotene as it is expected that -carotene is used Staphylococcus aureus, yeasts and molds in peanut butter can be up in the process of inhibition of peroxide formation. -carotene β β detrimental to human health [53,54]. In the present study (Table binds with the free radicals and blocks oxygen uptake during β 4), all samples tested negative for yeasts and molds and E. coli. oxidation and it is depleted as it binds with the free radicals [37]. However, Treatments 1, 2 and 3 tested positive for presence of S. This phenomenon explains why -carotene correlates negatively aureus and C0 tested negative (Table 3). The S. aureus ranged from with the PV and may also explain why treatments with high OFSP β 5.1*100cfu/g to 4*101cfu/g with treatment 3 recording the highest registered lower values of PV since -carotene inhibited the and treatment 2 had the least. The counts of S. aureus in treated formation of peroxides β peanut butter decreased with storage time. Table 4: Changes in colony counts for microorganisms in peanut butter with OFSP and control sample during storage.

Control (C0)cfu/g Treatment 1cfu/g Treatment 2cfu/g Treatment 3cfu/g

1st month

Yeasts and molds N. D N. D N. D N. D

S. aureus N. D 1×101 1.5×101 4×101

Coliforms N.D N. D N. D N. D

2nd month

Yeasts and molds N. D N. D N. D N. D

S. aureus N. D 1.2×100 6×100 3.4×101

Coliforms N.D N. D N. D N. D

3rd month Yeasts and molds N. D N. D N. D N. D S. aureus N. D N. D 4.35×100 1.7×101 Coliforms N. D N. D N. D N. D 4th month Yeasts and molds ND ND ND ND S. aureus ND ND 2.12×100 1×101 Coliforms ND ND ND ND

5th month

Yeasts and molds ND ND ND ND

S. aureus ND ND ND 5.1×100

Coliforms ND ND ND ND

N. D= Not Detected

Note: Control sample (C0), treatment 1 (5% OFSP flour), treatment 2 (10%OFSP flour) and treatment 3 (15% OFSP flour).

The bacteria S.aureus has several strains, and some are known for causing food spoilage which doesn’t result into harm to the that the peanut butter produced is safe for consumption since the et al. [58] in peanut butter is ˂103cfu/g of sample which also shows consumers but leads to food wasting (Institute of Food Technologists results from microbial analysis reported absence of yeasts and and Food and Drug Administration [55]. The production of S. aureus moulds. toxins is favored by minimum water activity (aw) of 0.9 [56], yet the Changes in sensory attributes of peanut butter with peanut butter is known to have very low water activity of below 0.7 storage time [56], which does not support production of toxins.

The bacteria S.aureus competes poorly in most foods with low moisture content [56,57], and owing to the fact the samples had No significant changes in color were noticed in all the samples moisture in the range of 1.8 to 2% which is far below the required (Table 5). Although significant changes in aroma, spread ability, in samples with storage time; the sensory scores were within for growth S. aureus and toxin production. This may also explain oiliness, taste, flavor and overall acceptability were noticed desirable range of 6 to 7and according to sensory scale 6 represents the reduction trend of Staphylococci numbers in the peanut butter like moderately and 7 like much (Table 5). The sensory attributes samples with storage time. are mainly affected by the changes in the fat quality of the peanut According to USDA (2010) set the minimum Coliform content to butter products due to fat oxidation [7,59]. However, the effect of be below 3.6cfu/g and all samples were free of E. coli. This indicates fat oxidation was not noticed in the OFSP enriched peanut butter good hygiene since the presence of coliform (E. coli) in peanut samples except in the control (C0). In the present study, microbial testing was done prior to sensory evaluation [60-62] and all treatments were found to be microbiologically safe for sensory butter can reflect the possibility of fecal contamination as coliforms animals [52]. The set standard for the yeasts and moulds by UNBS evaluation. are considered normal flora of the intestinal tract of humans and Table 5: Sensory changes for the control sample and peanut butter with added OFSP with storage time.

Color Aroma Oiliness Spreadability Taste Flavor Overall acceptability Control (C0) Month 1 6.68±1.65a 6.47±1.51a 6.08±1.89b 5.22±2.02b 6.35±2.20a 6.02±1.88b 6.22±1.82b Month 3 6.89±1.25a 6.39±1.33b 6.31±1.43b 5.47±1.94b 6.16±1.37b 6.47±1.20b 6.37±1.29b Month 5 7.14±1.48a 6.25±1.87b 6.22±1.85a 6.02±1.87b 6.20±1.55b 6.25±1.42b 6.50±1.44b Treatment 1 Month 1 7.31±1.29a 6.89±1.27a 6.85±1.59a 6.60±1.98a 6.79±1.55a 6.58±1.36b 7.20±1.18a Month 3 7.12±1.28a 6.58±1.48b 7.00±1.22a 6.79±1.55a 6.81±1.55a 6.81±1.21b 7.12±1.16a Month 5 7.45±1.27a 6.60±1.44b 6.83±1.54a 6.25±2.03a 6.97±1.45a 6.75±1.57a 7.06±1.26a Treatment 2 Month 1 7.41±1.06a 6.81±1.40a 6.87±1.24a 5.77±2.07b 6.89±1.60a 6.70±1.58a 7.04±1.58a Month 3 6.97±1.17a 6.14±1.23ab 6.10±1.46b 5.54±1.80b 6.43±1.51b 6.41±1.26b 6.47±1.23b Month 5 7.14±1.48a 6.60±1.42b 6.52±1.54b 5.85±1.81b 6.83±1.62a 6.91±1.2a 6.87±1.10b Treatment 3 Month 1 6.83±1.98a 6.25±1.65a 6.27±1.84a 6.25±2.03a 6.22±1.83a 6.06±1.82b 6.33±1.95a Month 3 7.35±1.31a 6.81±1.51b 6.72±1.46a 6.00±1.83b 6.89±1.77a 6.81±1.37a 6.91±1.47a Month 5 7.58±1.04a 7.10±1.11b 7.00±1.48a 6.68±1.96a 7.16±1.11a 7.29±0.98a 7.43±1.16a

All values represent means ±SD; Values with same letter in a column are not significantly different (p≤0.05).

Control sample (C0), Treatment 1: (5% OFSP flour), Treatment 2: (10%OFSP flour) and Treatment 3: (15% OFSP flour).

Conclusion that OFSP can be used in peanut butter to enhance its nutritional value (vitamin A requirements) of the school-going children. There is a need to encourage the diverse utilization of OFSP in peanut peanut butter improved -carotene content, which increases with The findings suggest that use of OFSP in the production of butter production to improve the vitamin A status of school going high substitution levels. Treatment 3 with 15% OFSP had the β children. This could be one of the most possible ways of improving highest -carotene, highest beta-carotene retention on shelf, better OFSP utilization by incorporating it in common local products like fat quality and had acceptable sensory score. Thus, it is concluded β Oddi.

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